Tumors are able to actively create tolerance to their own antigens. This acquired tolerance includes both anergy and potent Treg-mediated immunosuppression, and represents a fundamental barrier to the immunotherapy of cancer. The current proposal focuses on the tolerance-inducing enzyme indoleamine 2,3-dioxygenase (IDO), which functions as a key molecular mechanism linking two potent tolerogenic pathways in tumor-draining lymph nodes: IDO-expressing immunoregulatory DCs, and regulatory T cells (Tregs). The proposal will examine the role of IDO in the critical window of time during recovery from cytotoxic chemotherapy. Chemotherapy releases a wave of tumor antigens and transiently perturbs tolerance. Preliminary data show that IDO is a key molecular mechanism by which the tumor re-establishes tolerance to itself following chemotherapy. IDO acts both by directly anergizing tumor- specific CDS" effector T cells, and by markedly activating the suppressor function of pre-existing Foxp3+ Tregs. In the post-chemotherapy period, IDO-dependent tolerogenic mechanisms are selectively preserved at high levels in tumor-draining lymph nodes. The hypothesis underlying the proposal is thus that IDO represents a key molecular mechanism required to maintain tolerance to tumor antigens following chemotherapy. The overall goal of the proposal is to develop mechanistically-based, clinically applicable strategies to break tolerance to tumor antigens, based on the combination of IDO-inhibitor drugs and chemotherapy. Aim 1 will test the hypothesis that, prior to chemotherapy, the tumor-draining lymph node drives acquired systemic tolerance to tumor antigens via the synergistic combination of IDO+ pDCs and activated Tregs. Aim 2 will test the hypothesis that chemotherapy is able to transiently disrupt tolerance to established tumors, but only if the IDO pathway is blocked during the post-chemotherapy recovery period. Aim 3 will test the hypothesis that the window of disrupted tolerance opened by the combination of IDO-inhibition plus chemotherapy will allow curative anti-tumor T cell responses to be generated by active immunotherapy delivered in this window. The significance of the current proposal is that it aims to develop clinically-applicable treatment regimens for breaking tolerance to tumors, based on a strong and novel mechanistic rationale for synergy between chemotherapy and IDO-inhibitor drugs. Since a number of vaccines and immunomodulators are currently limited by the barrier of tumor-induced tolerance, a tolerance-breaking regimen based on IDO-inhibitors plus chemotherapy has the potential to enhance the efficacy of a variety of existing immunotherapy strategies.